The discovery of novel and useful materials depends largely on the capacity to make and characterize new compositions of matter. As a result, recent research relating to novel materials that have useful biological, chemical, and/or physical properties has focused on the development and implementation of new methods and systems for synthesizing and evaluating potentially useful chemical compounds. In particular, high-speed combinatorial methods have been developed to address the general need in the art for systematic, efficient, and economical material synthesis techniques, as well as for methods to analyze and to screen novel materials for useful properties.
Generally, it is important to control the quality of the starting materials in any chemical synthesis process. Otherwise, the integrity of the process and the quality of the resulting product are compromised. Quality control of the starting materials is a particularly important issue in combinatorial synthesis procedures. In such procedures, a large number of starting compounds may be dispensed in a predetermined sequence from a compound library to synthesize, for example, a batch of a drug. As a further example, such procedures may be used in peptide drug discovery applications to synthesize a drug containing a specific peptide sequence. Should any of the starting compounds contain an unacceptable level of a contaminant or exhibit an unacceptable degree of degradation, the synthesized compound may be rendered useless. In effect, all starting compounds employed for the batch synthesis would be wasted. This is particularly problematic when one or more of the starting compounds are rare or expensive.
Similarly, combinatorial testing techniques may be employed in analytical and testing procedures. For example, a combination of two or more pharmacologically active candidate compounds may be delivered to a test sample in order to assess whether synergistic effects are achieved. If any one of the candidate compounds is compromised in quality, however, the accuracy and reliability of the assessment may be reduced. Thus, further testing may be necessary, adding significantly to the overall time and cost associated with the combinatorial testing process.
High-speed combinatorial methods often involve the use of array technologies that require accurate dispensing of fluids, each having a precisely known chemical composition, concentration, stoichiometry, ratio of reagents, and/or volume. Such array technologies may be employed to carry out various synthetic processes and evaluations. Array technologies may use large numbers of different fluids to form a plurality of reservoirs that, when arranged appropriately, create combinatorial libraries. In order to carry out combinatorial methods, a number of fluid dispensing techniques have been explored, such as pin spotting, pipetting, inkjet printing, and acoustic ejection. Many of these techniques possess inherent drawbacks that must be addressed, however, before the fluid dispensing accuracy required for the combinatorial methods can be achieved. For instance, a number of fluid dispensing systems are constructed using networks of tubing or other fluid-transporting vessels. Tubing, in particular, can entrap air bubbles, and nozzles may become clogged by lodged particulates. As a result, system failure may occur and cause spurious results. Furthermore, cross-contamination between the reservoirs of compound libraries may occur due to inadequate flushing of tubing and pipette tips between fluid transfer events. Cross-contamination can easily lead to inaccurate and misleading results.
Acoustic ejection provides a number of advantages over other fluid dispensing technologies. In contrast to inkjet devices, nozzleless fluid ejection devices are not subject to clogging and their associated disadvantages, e.g., misdirected fluid or improperly sized droplets. Furthermore, acoustic technology does not require the use of tubing or involve invasive mechanical actions, for example, those associated with the introduction of a pipette tip into a reservoir of fluid.
Acoustic ejection has been described in a number of patents. For example, U.S. Pat. No. 4,308,547 to Lovelady et al. describes a liquid drop emitter that utilizes acoustic principles to eject droplets from a body of liquid onto a moving document to result in the formation of characters or barcodes thereon. A nozzleless inkjet printing apparatus is used such that controlled drops of ink are propelled by an acoustical force produced by a curved transducer at or below the surface of the ink. Similarly, U.S. Pat. No. 6,666,541 to Ellson et al. describes a device for acoustically ejecting a plurality of fluid droplets toward discrete sites on a substrate surface for deposition thereon. The device includes an acoustic radiation generator that may be used to eject fluid droplets from a reservoir, as well as to produce a detection acoustic wave that is transmitted to the fluid surface of the reservoir to become a reflected acoustic wave. Characteristics of the reflected acoustic radiation may then be analyzed in order to assess the spatial relationship between the acoustic radiation generator and the fluid surface. Thus, acoustic ejection may provide an added advantage in that the proper use of acoustic radiation provides feedback relating to the process of acoustic ejection itself.
Regardless of the dispensing technique used, however, inventory and materials handling limitations generally dictate the capacity of combinatorial methods to synthesize and analyze increasing numbers of sample materials. For instance, during the formatting and dispensing processes, microplates that contain a plurality of fluids in individual wells may be thawed, and the contents of selected wells can then be extracted for use in a combinatorial method. When a pipetting system is employed during extraction, a minimum loading volume may be required for the system to function properly. Similarly, other fluid dispensing systems may also require a certain minimum reservoir volume to function properly. Thus, for any fluid dispensing system, it is important to monitor the reservoir contents to ensure that at least a minimum amount of fluid is provided. Such content monitoring generally serves to indicate the overall performance of a fluid dispensing system, as well as to maintain the integrity of the combinatorial methods.
During combinatorial synthesis or analysis processes, environmental effects may play a role in altering the contents of reservoirs on a well plate or microplate. For example, dimethylsulfoxide (DMSO) is an organic solvent commonly employed to dissolve or suspend many of the compounds found in drug libraries. DMSO is highly hygroscopic and tends to absorb ambient water with which it comes into contact. In turn, the absorption of water dilutes the concentration the compounds as well as alters the ability of the DMSO to dissolve or suspend the compounds. Furthermore, the absorption of water may promote the decomposition of water-sensitive compounds.
Acoustic monitoring makes it possible to determine the DMSO content of DMSO/water mixtures in microplate reservoirs, for DMSO concentrations ranging from about 70 to 100% by volume. When the fluid in the microplate well is a binary mixture of DMSO and water, adding water to or removing water from the mixture results in a change in the echo amplitude from the fluid/well-bottom interface. Measurement of the amplitude of reflection from microplate surfaces enables a calculation of acoustic impedance for the liquid in the microplate well. When acoustic impedance is translated to DMSO concentration in a DMSO/water mixture by the appropriate empirical model, the results are accurate to within a few percentage points at room temperature.
However, if the microplate temperature deviates from room temperature, both the acoustic impedance of the microplate, and potentially that of the fluid in the wells, will change. As a consequence, the change in the amplitude of reflection for acoustic energy at the fluid/well interface will be interpreted as a change in DMSO/water composition.
For example, if a microplate containing a frozen DMSO/water mixture is processed by an acoustic instrument, e.g., the Echo 550 instrument (Labcyte Inc., Sunnyvale, Calif.), the initial DMSO content is incorrectly measured as being less than 40%. It takes nearly one hour before the microplate warms to room temperature, when the equilibrium acoustic impedances needed for the room temperature empirical model described above are reached, so that the composition of the DMSO/water mixtures in the wells can be accurately measured.
Benefits in accuracy and in time used would be obtained if microplates did not have to be at thermal equilibrium in order to accurately measure fluid composition in a well. A device and method is needed for making accurate compositional measurements without having to bring well plates to room temperature, by compensating for thermal variations in acoustic impedance and changes in the resulting echoes.
A number of patents describe the use of acoustic energy to assess the contents of a container. U.S. Pat. No. 5,507,178 to Dam, for example, describes a sensor for determining the presence of a liquid and for identifying the type of liquid in a container. The ultrasonic sensor determines the presence of the liquid through an ultrasonic liquid presence sensing means and identifies the type of liquid through a liquid type identification means, and includes a pair of electrodes and an electrical pulse generating means. This device suffers from the disadvantage that the sensor must be placed in contact with the liquid.
U.S. Pat. No. 5,880,364 to Dam, on the other hand, describes a non-contact ultrasonic system for measuring the volume of liquid in a plurality of containers. An ultrasonic sensor is disposed opposite to the tops of the containers. A narrow beam of ultrasonic radiation is transmitted from the sensor to the open top of an opposing container, and is then reflected from the air-liquid interface of the container back to the sensor. By using the round trip transit time of the radiation and the dimensions of the containers being measured, the volume of liquid in the container can be calculated. This device cannot be used to assess the contents of sealed containers. In addition, the device lacks precision because air is a poor conductor of acoustic energy. Thus, while this device may provide rough estimates of the volumes of liquid in relatively large containers, it is unsuitable for use in providing a detailed assessment of the contents of reservoirs typically used in combinatorial techniques. In particular, this device cannot determine the position of the bottom of containers, since substantially all of the emitted acoustic energy is reflected from the liquid surface and does not penetrate to detect the bottom. Low volume reservoirs in microplates are regular arrays of fluid containers, and the location of the bottoms of the containers can vary by a significant fraction of the nominal height of a container due to distortions in the plate, such as bowing. Thus, detection of only the position of the liquid surface leads to significant errors in height and thus volume estimation in common containers.
There is thus a need in the art for improved methods and devices that are capable of determining temperature-dependent properties of reservoirs and fluids contained therein. Such capabilities would be particularly useful for tracking the quality of compound libraries in solution, and the quantities of the compounds therein, for combinatorial and screening applications. In particular, it would be useful to determine such properties for a range of reservoir and microplate temperatures.